The family of ?-aminobutyric acid type A receptors (GABA(A)Rs) are the major inhibitory neuro? receptors in the human brain. Some two dozen isoforms, each made up of five subunits selected from the 19 established subunits, mediate many different physiological pathways associated with consciousness, sedation, anxiety, epilepsy, depression and postpartum depression. Mutations in the GABA(A)R subunits are associated with diseases such as epilepsy and schizophrenia. They are also the target of many drugs that for example reversibly suspend conciousness or seek to control epilepsy or anxiety without inducing sedation. Our overall long-term goal is to establish the relationship between the structure of synaptic and extrasynaptic GABA(A)R and how they function. The basic strategy, established in two recent papers, is to use single particle cryo-EM to determine structures of full-length, glycosylated human GABA(A)Rs, purified and reconstituted in a state that preserves conformation changes and to use the same receptors to study structure and function in parallel. This is possible because of the development of inducible, high yielding stable cell lines. During activation and deactivation, GABA(A)Rs undergo conformation changes that open and close allosteric sites some of which are used by endogenous ligands (e.g. neurosteroids) and other by drugs (e.g. general anesthetics, benzodiazepines). These allosteric ligands, discovered serendipitously, provide clues to the existence of such sites. We aim to provide a basic understanding of how the structure of these allosteric pockets depends on a receptor?s conformation. This will provide the basic mechanistic knowledge that will enable others to develop ways to control GABA(A)R function. Our working hypothesis is that allosteric sites located between subunits in the TMD can accommodate positive and negative allosteric modulators (PAMs and NAMs respectively) as well as null allosteric ligands (NALs), just as benzodiazepines can in the ?+/?? interface in the ECD. Our first specific aim concerns extrasynaptic receptors, which do not contain a benzodiazepine site. Here the basic information on stoichiometry and subunit arrangement is lacking, and this problem must be addressed first. Two ligands, DS2 and ketamine, both selective for ?-subunit?containing extrasynaptic receptors, provide clues to the existence of unique allosteric sites, perhaps in contact with the ?-subunit, that open up during activation. The second specific aim tests the hypothesis that agents binding in the TMD of synaptic receptors can act as PAMs, NAMs and NALs. PAMs are well established, and this aim focuses on NAMs and NALs. Based on existing structures in several conformations, we will design and synthesize NAMs and NALs with the goal of obtaining ligands suitable for structural determination that will improve the mechanistic understanding of GABA(A)R function. The significance is that this may lead to the development of anesthetic antagonists (NALs) and sedative (partial NAMs), as well as a deeper understanding of the mechanisms of GABA(A)R function.